Preparation for improving immune response and application thereof

Abstract

The invention relates to a preparation for improving immune response and application thereof, and relates to the technical field of immune preparations. The preparation comprises at least one of isopentenol and 4-hydroxyphenylpyruvic acid. The application comprises an application of the preparation and a vaccine in combined immunization, an application of the preparation as an adjuvant in preparation of a composite vaccine, and an application of the preparation in preparation of an anti-tumor product. The application of the isopentenol and / or 4-hydroxyphenylpyruvic acid as the biomarker comprises the application in monitoring the immune response level of an organism and the application in screening medicines for regulating metabolic pathways.

Description

Technical Field

[0001] The present disclosure relates to the technical field of immune preparations, and in particular, to a preparation for improving immune response and application thereof. Background Art

[0002] A survey of people who have received the inactivated COVID-19 vaccine found that different individuals have different immune response activities to the vaccine, indicating that there may be individual factors that affect the body's immune response to vaccination.

[0003] Nutritional metabolism provides the material basis for the activities of immune cells by providing the nutrients required by cells, such as amino acids, lipids, vitamins and trace elements. Studies have found that the role of these nutrients is far more than that. For example, vitamin A is involved in the control and function of the immune system. It not only affects development, but also participates in the function of innate immunity or adaptive immune cells. Through its metabolite retinoic acid, it binds to the ligand of nuclear receptors to control the transcription and expression of target genes, linking the microenvironment changes of immune cells to their functions. Arginine and tryptophan are believed to be able to regulate the activity and anti-inflammatory response of immune cells. Metabolites, as signaling molecules, affect the function and response of immune cells and provide cells with clues to respond to changes in the microenvironment. The mTOR-phosphoinositide 3kinase (PI3K) classical signaling pathway in adaptive immunity is a typical example. This phenomenon is more common in tumor metabolism. After immune cells sense small molecule metabolite signals in the environment, they undergo metabolic reprogramming in cell proliferation, differentiation and execution of functional effects, which is crucial for regulating immune function responses.

[0004] The impact of metabolism on immunity mainly comes from three aspects: First, the proliferation and differentiation of immune cells and the production of antibodies in the immune response process require a lot of energy, which is provided by the aerobic or anaerobic metabolism of cells. Secondly, nutritional metabolism provides the nutrients necessary for the body to synthesize antibodies, and some trace elements such as vitamins will also affect the response of immune cells. Finally, small molecule metabolites produced in metabolic pathways can have signal regulation effects and affect the function of immune cells. Therefore, studying the abundance of serum metabolites in the population and the changes in metabolites during the immune response process can provide a more comprehensive understanding of the role of metabolites in the immune response, screen metabolites that enhance the immune response, and then provide methods to enhance the immune response.

[0005] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute the prior art known to ordinary technicians in the field. Summary of the invention

[0006] The purpose of the present disclosure is to overcome the deficiencies of the above-mentioned prior art and provide a preparation for improving immune response and its application, which can improve vaccine immune response and anti-tumor therapeutic effect by comprising a preparation of isopentenol and 4-hydroxyphenylpyruvic acid.

[0007] According to a first aspect of the present disclosure, a preparation for improving an immune response is provided, the preparation comprising at least one of prenol and 4-hydroxyphenylpyruvic acid.

[0008] In an exemplary embodiment of the present disclosure, the preparation is an injection or an oral preparation.

[0009] In an exemplary embodiment of the present disclosure, the isopentenol includes 3-methylbut-1-en-1-ol and 3-methylbut-2-en-1-ol.

[0010] According to a second aspect of the present disclosure, there is provided use of the above-mentioned preparation in the preparation of a combined immune product; the combined immune product comprises the preparation and a vaccine.

[0011] In an exemplary embodiment of the present disclosure, the combined immunization product achieves combined immunization by the following method: S10, obtaining a combined immunization product, the combined immunization product comprising the preparation and the vaccine;

[0012] S20, administration before vaccine immunization: before vaccine immunization, the first preset process is used to complete the administration of the preparation described in step S10;

[0013] Administration during vaccine immunization: the administration of the vaccine described in step S10 is completed by using the second preset process, and at the same time, the administration of the preparation described in step S10 is completed by using the third preset process during the vaccine immunization process;

[0014] Administration after vaccine immunization: After the last vaccine immunization, the fourth preset process is used to complete the administration of the preparation described in step S10.

[0015] According to a third aspect of the present disclosure, there is provided use of the above-mentioned preparation as an adjuvant in the preparation of a composite vaccine.

[0016] In an exemplary embodiment of the present disclosure, the immunization method of the composite vaccine comprises the following steps:

[0017] S1. Obtaining a composite vaccine, wherein the composite vaccine comprises a vaccine and the preparation;

[0018] S2. Complete the immunization administration of the combined vaccine according to the fifth preset process.

[0019] According to a fourth aspect of the present disclosure, there is provided use of the above-mentioned preparation in the preparation of an anti-tumor product.

[0020] In an exemplary embodiment of the present disclosure, the anti-tumor product is an anti-tumor compound preparation or a combined anti-tumor composition;

[0021] The composite preparation comprises the preparation and an anti-tumor drug, and is used to allow the preparation and the anti-tumor drug to be administered simultaneously;

[0022] The combined anti-tumor composition comprises the preparation and an anti-tumor drug, and is used to allow the preparation and the anti-tumor drug to be used in combination and administered separately.

[0023] According to a fifth aspect of the present disclosure, there is provided the use of isopentenol and / or 4-hydroxyphenylpyruvate as biomarkers in monitoring the level of immune response of an organism.

[0024] According to a sixth aspect of the present disclosure, there is provided use of isopentenol and / or 4-hydroxyphenylpyruvate as biomarkers in screening drugs that regulate metabolic pathways;

[0025] The drug for regulating metabolic pathways is used to increase the synthesis of isopentenol and / or 4-hydroxyphenylpyruvate, or the drug for regulating metabolic pathways is used to reduce the degradation or excretion of isopentenol and / or 4-hydroxyphenylpyruvate.

[0026] The present disclosure provides a preparation for improving immune response. On the one hand, the preparation can be used as a combined drug or vaccine adjuvant for improving immune response in a vaccine to improve the body's immune response to the vaccine and improve the immune prevention effect of the vaccine. On the other hand, the preparation can also be used to prepare anti-tumor products, improve the body's anti-tumor immune response, and then eliminate tumor cells expressing tumor-specific antigens to achieve a tumor treatment effect.

[0027] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings herein are incorporated into the specification and constitute a part of the specification, illustrate embodiments consistent with the present disclosure, and together with the specification are used to explain the principles of the present disclosure. Obviously, the accompanying drawings described below are only some embodiments of the present disclosure, and for ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without creative work.

[0029] Figure 1 This is a schematic diagram of the sVNT titer results of serum antibodies of participants on days 28, 90 and 300 after the third immunization in one embodiment of the present disclosure.

[0030] Figure 2This is a graph of the serum antibody decay curve from the 28th day to the 300th day after the third immunization of a participant in one embodiment of the present disclosure.

[0031] Figure 3 This is a schematic diagram of the results of differential metabolites in the serum of the high antibody group and the low antibody group on the 28th day after the third immunization of the subjects in one embodiment of the present disclosure.

[0032] Figure 4 This is a schematic diagram of the results of differential metabolites in the serum of the high antibody group and the low antibody group on the 90th day after the third immunization of the subjects in one embodiment of the present disclosure.

[0033] Figure 5 This is a schematic diagram comparing the abundance of 4-hydroxyphenylpyruvate in the serum of the high antibody group and the low antibody group on the 28th and 90th day after the third immunization of the subjects in one embodiment of the present disclosure.

[0034] Figure 6 This is a schematic diagram comparing the abundance of isopentenol in the serum of the high antibody group and the low antibody group on the 28th and 90th days after the third immunization of the subjects in one embodiment of the present disclosure.

[0035] Figure 7 This is a schematic diagram of the change trend of serum abundance of isopentenol in each subject in the high antibody group and the low antibody group on the 28th day and the 90th day after the third immunization of the subject in one embodiment of the present disclosure.

[0036] Figure 8 This is a schematic diagram of the change trend of serum abundance of 4-hydroxyphenylpyruvate in each subject in the high antibody group and the low antibody group on the 28th day and the 90th day after the third immunization of the subject in one embodiment of the present disclosure.

[0037] Fig. 9 Schematic diagram of the pathway for the production of coenzyme Q10 from prenol and 4-hydroxyphenylpyruvate.

[0038] Fig.10 The present invention is an embodiment of the present invention, in which serum prenol and 4-HPP are used to evaluate the efficacy of immune response after immunization.

[0039] Fig.11 This is a schematic diagram of the results of the correlation analysis between serum APO-A1 concentration and serum isopentenol abundance on the 28th day after the third immunization of the subject in one embodiment of the present disclosure.

[0040] Fig.12This is a schematic diagram showing the comparison of sVNT titers of COVID-19 antibodies in the serum of mice in the inactivated vaccine + metabolite group, inactivated vaccine group, metabolite group and control group on the 42nd day after the first immunization of mice in one embodiment of the present disclosure.

[0041] Fig.13 This is a schematic diagram comparing the serum APO-A1 concentrations of mice in the inactivated vaccine + metabolite group, inactivated vaccine group, metabolite group and control group on the 42nd day after the first immunization of mice in one embodiment of the present disclosure.

[0042] Fig.14 This is a schematic diagram of the comparison of tumor volume growth curves of mice in the metabolic group, anti-PD1 antibody treatment group, metabolite combined with anti-PD1 antibody treatment group and control group from day 0 to day 24 of tumor-bearing in one embodiment of the present invention. DETAILED DESCRIPTION

[0043] Example embodiments will now be described more fully with reference to the accompanying drawings. However, example embodiments can be implemented in many forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be comprehensive and complete and will fully convey the concept of the example embodiments to those skilled in the art.

[0044] The term "vaccine" refers to a biological product that uses pathogenic microorganisms or their components or metabolites as starting materials, is prepared using biotechnology, and is used to prevent and treat corresponding human diseases.

[0045] The term "tumor therapeutic vaccine" refers to a class of products that achieve therapeutic effects such as inhibiting or killing tumor cells, clearing tiny residual lesions or precancerous lesions, and establishing lasting anti-tumor memory by inducing or enhancing the body's specific active immune response to tumor antigens. It injects tumor antigens into the body in different forms, and after being recognized, ingested, and processed by antigen-presenting cells, the antigen information is presented on the cell surface, inducing the body to produce antigen-specific immune responses, especially the activation of cytotoxic T lymphocytes, which then migrate and infiltrate into the tumor microenvironment to play a role in killing tumor cells. Its mechanism of action is different from that of tumor therapeutic vaccines (such as BCG) with non-specific immune effects. The key component of tumor therapeutic vaccines is tumor antigens, which can be divided into two categories according to tumor specificity: tumor-associated antigen (TAA) and tumor-specific antigen (TSA). Tumor therapeutic vaccines are diverse in form, including but not limited to cell vector vaccines, viral vector vaccines, peptide vaccines, protein vaccines, and nucleic acid vaccines.

[0046] The term "composite vaccine" in the present disclosure refers to a vaccine comprising the formulation provided by the present disclosure, to distinguish it from existing or commercially available vaccines. That is, if the formulation of the present disclosure is used as an adjuvant and a vaccine to prepare a composite vaccine, it does not exclude the possibility that the original vaccine contains other adjuvants or other added ingredients.

[0047] Unless otherwise defined, all terms and phrases used herein include the meanings that the terms and phrases have in the art unless clearly indicated to the contrary or clearly apparent from the context in which the terms and phrases are used.

[0048] The present disclosure provides a preparation for improving immune response, the preparation comprising at least one of prenol and 4-hydroxyphenylpyruvate. That is, the preparation may only contain prenol, or the preparation may only contain 4-hydroxyphenylpyruvate (4-HPP), or the preparation may simultaneously contain prenol and 4-hydroxyphenylpyruvate.

[0049] In one embodiment of the present disclosure, the preparation can be an injection or an oral preparation. Oral administration has a high acceptance rate among the vaccination population. Compared with oral administration, the preparation can be injected for immunization, which can more effectively improve the immune response.

[0050] In one embodiment of the present disclosure, the isopentenol includes 3-methylbut-1-en-1-ol and 3-methylbut-2-en-1-ol.

[0051] The disclosed embodiments also provide the use of the above-mentioned preparation in the preparation of a combined immune product; the combined immune product comprises the above-mentioned preparation and a vaccine.

[0052] In one example, the combined immunization product achieves combined immunization by the following method:

[0053] S10. Obtaining a combined immune product, wherein the combined immune product comprises the preparation and the vaccine;

[0054] S20, administration before vaccine immunization: before vaccine immunization, the first preset process is adopted to complete the administration of the preparation described in step S10. Before vaccine immunization, supplementing the body with exogenous isopentenol and / or 4-hydroxyphenylpyruvic acid can promote the large-scale generation of coenzyme Q10 and prepare for the antigen presentation of APCs after vaccine immunization. Further, the first preset process can select a suitable immunization cycle, immunization interval and immunization dose according to the metabolic cycle of the preparation prepared by isopentenol and / or 4-HPP for different populations, and the combined effect of the preparation and the vaccine in the immune response. Exemplarily, a suitable administration time before vaccine immunization can be selected according to the metabolic curve of the preparation for people of different age groups. In this way, the peak blood concentration of isopentenol or 4-HPP in the preparation can be consistent with the time of vaccine immunization, and the blood concentration of isopentenol or 4-HPP in the preparation can be maintained above the concentration that can improve the immune response, so as to improve the immune response level of the vaccine through the preparation.

[0055] Administration during vaccine immunization: The second preset process is used to complete the administration of the vaccine described in step S10, and at the same time, the third preset process is used to complete the administration of the preparation described in step S10 during the vaccine immunization process. During the vaccine immunization process, exogenous isopentenol and / or 4-hydroxyphenylpyruvic acid are continuously supplemented to the body to promote the antigen presentation process of APCs, thereby increasing the antibody level in the serum and improving the effect of the vaccine in preventing diseases.

[0056] Exemplarily, the second preset process can be carried out according to the vaccination cycle, vaccination interval and vaccination dose of the vaccine itself, or according to the combined effect of the vaccine and the preparation in the immune response, a suitable vaccination cycle, vaccination interval and vaccination dose can be selected. It is worth noting that, based on the combined immunization with the preparation, the second preset process corresponding to different vaccines may be different.

[0057] Exemplarily, the third preset process can select a suitable vaccination cycle, vaccination interval and vaccination dose according to the metabolism of the preparation in the body, so that the blood concentration of prenol or 4-HPP in the preparation is maintained above the effective concentration that can improve the immune response. The third preset process can be the same as the first preset process or different.

[0058] Administration after vaccine immunization: After the last vaccine immunization, the fourth preset process is used to complete the administration of the preparation described in step S10. After the vaccine immunization is completed, exogenous isopentenol and / or 4-hydroxyphenylpyruvic acid are continued to be supplemented to prolong the time of high-level immune response, form a stable immune response level in the body (for example, so that the serum antibody titer remains in a relatively stable state), and improve the protective effect of the vaccine on the body. The fourth preset process can be the same as the third preset process, or it can be different.

[0059] This embodiment also provides the use of the above preparation as an adjuvant in the preparation of a composite vaccine.

[0060] In one example, the composite vaccine comprises a vaccine and the preparation, that is, the vaccine and the preparation are prepared together into a new composite vaccine for immunization administration.

[0061] In one example, the immunization method of the composite vaccine comprises the following steps:

[0062] S1. Obtaining a composite vaccine, wherein the composite vaccine comprises a vaccine and the preparation;

[0063] S2. Complete the immunization administration of the composite vaccine according to the fifth preset process. Further, the fifth preset process can be carried out according to the immunization cycle, immunization interval and immunization dose of the vaccine itself, or according to the combined effect of the vaccine and the preparation in the immune response, select a suitable immunization cycle, immunization interval and immunization dose. In other words, after different vaccines and the preparation form a composite vaccine, the corresponding fifth preset process may be different.

[0064] This embodiment also provides the use of the above preparation in the preparation of anti-tumor products.

[0065] In one embodiment of the present disclosure, the anti-tumor product may only include the preparation. Exemplarily, the preparation is prepared into an anti-tumor drug, which can be combined with other tumor treatment methods for anti-tumor treatment. For example, it can be used in combination with chemical anti-tumor therapy, radiotherapy, traditional Chinese medicine anti-tumor therapy, etc.

[0066] In one embodiment of the present disclosure, the anti-tumor product is an anti-tumor compound preparation or a combined anti-tumor composition. The anti-tumor compound preparation includes the preparation and the anti-tumor drug, and is used to allow the preparation and the anti-tumor drug to be administered simultaneously. That is, in the anti-tumor compound preparation, the preparation and the anti-tumor drug can be mixed in an appropriate ratio to prepare a certain dosage form.

[0067] The combined anti-tumor composition comprises the preparation and the anti-tumor drug, and is used to make the preparation and the anti-tumor drug used in combination and administered separately. That is, in the combined anti-tumor composition, the preparation and the anti-tumor drug can be administered in sequence according to actual needs.

[0068] The anti-tumor drug can be a tumor therapeutic vaccine that inhibits or kills tumor cells through an immune response, or it can be an immune checkpoint inhibitor, or it can be a chemical anti-tumor drug, an anti-tumor prescription containing traditional Chinese medicine, or an anti-tumor Chinese patent medicine.

[0069] This embodiment also provides the use of isopentenol and / or 4-HPP as biomarkers in monitoring the state of immune response of the body. Exemplarily, by monitoring the changes in the levels of serum isopentenol and / or 4-HPP during immunotherapy, the level of immune response in the body is evaluated and the prognosis of treatment is inferred.

[0070] The present embodiment also provides the application of prenol and / or 4-HPP as biomarkers in screening the medicine for regulating metabolic pathways. The medicine for regulating metabolic pathways is used to improve the synthesis of prenol and / or 4-HPP, or the medicine for regulating metabolic pathways is used to reduce the degradation or discharge of prenol and / or 4-HPP. It is understandable that the experiment verifies that the level of prenol and / or 4-HPP that improves body can improve the immune response of body to vaccine. So, on the one hand, by supplementing the exogenous preparation, the prenol and / or 4-HPP generation of body itself can be improved or its loss can be reduced on the other hand. In this way, by detecting prenol and 4-HPP, screening can act on the medicine of the synthetic pathway of prenol and / or 4-HPP, and can also screen and act on the medicine that reduces the degradation or discharge pathway of prenol and / or 4-HPP.

[0071] The solution of the present disclosure is further described below in conjunction with specific embodiments.

[0072] Embodiment 1

[0073] In this example, a cohort population was followed up and blood samples were collected from the participants at least once to assess the serum COVID-19 antibody sVNT titer.

[0074] From November 1, 2021 to August 10, 2022, 2440 participants were recruited for cohort follow-up, excluding: ① 40 participants with no vaccination information; ② 20 participants whose vaccination type did not meet the BBIBP-CorV (Sinopharm Beijing Institute of Biological Products) or CornoVac (Beijing Sinovac) COVID-19 vaccine mentioned in the inclusion criteria; ③ 2 participants whose interval between the second and third doses of vaccination was less than 180 days. A total of 62 participants were excluded, and finally 2378 participants were included in the cohort.

[0075] like Figure 1 The COVID-19 antibody sVNT titers in the serum of participants at three time points, 28 days, 90 days, and 300 days after the third dose of vaccination, are shown, where n is the number of participants in the sampling at each sampling time point. Figure 1 It can be seen that the serum COVID-19 antibody sVNT titer on day 90 decreased by 2.42 times compared with day 28, the sVNT titer on day 300 decreased by 2.20 times compared with day 90, and the sVNT titer on day 300 decreased by 5.31 times compared with day 28. All 2378 participants in the cohort were included in the antibody decay analysis, and the smoothing spline method was used to fit the following Figure 2 The antibody decay curve shown in Figure 1 was used to verify the dynamic changes of serum COVID-19 antibody levels over time. The longitudinal analysis of the serum COVID-19 antibody sVNT titer decay curve within 300 days after three doses of vaccine showed that the sVNT titer decayed rapidly within 90 days after vaccination, while the decay rate from day 90 to day 300 was relatively slow.

[0076] This example shows that the serum antibody sVNT titer is at its peak within 28 days after the third dose of vaccination, and the antibody sVNT titer gradually decays from the 28th day to the 90th day, and enters a stable stage after the 90th day.

[0077] Embodiment 2

[0078] Non-targeted metabolomics coupled with liquid chromatography-mass spectrometry (LC-MS) was used to detect metabolites in serum samples to study the changes in metabolites in serum samples of each experimental population in different experimental groups.

[0079] 1. Experimental Grouping

[0080] Inclusion criteria: 1) Healthy people without comorbidities; 2) Three doses of inactivated COVID-19 vaccine (BBIBP-CorV from Beijing Institute of Biological Products of Sinopharm or Sinovac CoronaVac from Beijing Sinovac Biotech Co., Ltd.) were administered to the subjects. The average interval between the first and second doses of the vaccine was 20 days (range 18-22 days), and the interval between the second and third doses was greater than 180 days. 3) The serum sVNT titer in the serum samples collected on the 28th and 90th days after the third dose of the vaccine was ≥1024IU / mL or the sVNT titer was ≤512IU / mL. The serum sVNT titer is a surrogate virus neutralization test titer for serum samples.

[0081] That is to say, after the third dose of the vaccine, serum samples were collected on the 28th and 90th days for testing. Among them, the subjects who simultaneously met the serum sVNT titer ≥1024IU / mL on the 28th and 90th days were the high antibody group (experimental group), with a total of 81 subjects in the high antibody group; the subjects who simultaneously met the serum sVNT titer ≤512IU / mL on the 28th and 90th days were the low antibody group (control group), with a total of 80 subjects in the low antibody group. The basic information of each subject in the high antibody group (High group in Table 1) and the low antibody group (Low group in Table 1) is shown in Table 1.

[0082] Table 1. Basic information of subjects in the high antibody group and the low antibody group

[0083]

[0084] 2. Metabolomics detection and analysis

[0085] For subjects in the high antibody group and the low antibody group, 161 serum samples were collected on the 28th day after the third dose of vaccination, and 155 serum samples were collected on the 90th day (6 samples were unqualified and were eliminated) for non-targeted metabolomics detection. The obtained base peak chromatogram (BPC) was subjected to data preprocessing and qualitative and quantitative analysis, and a total of 15,991 compounds were detected. The detected compounds were analyzed using the Human Metabolome Database (HMDB), LIPIDMAPS database (version number: 2.3), METLIN database and tandem mass spectrometry database, and a total of 6,303 (39.4%) serum metabolites were identified.

[0086] The serum metabolites of the subjects obtained on the 28th day and the 90th day after the third dose of the vaccine were subjected to t-test (Student's t-test) and fold change (FC) analysis to compare the differences in serum metabolite levels between the two groups. According to the screening criteria of FC value ≥1.2 or ≤1 / 1.2 and p value <0.05, the differential metabolites (DEMs) at the 28th day and the 90th day were screened out. Figure 3 As shown in the figure, 327 DEMs were detected in the serum samples of the high antibody group and the low antibody group on the 28th day after the third dose of vaccination; among them, 152 DEMs were upregulated in the high antibody group relative to the low antibody group, while 175 DEMs were downregulated in the high antibody group relative to the low antibody group. Figure 4 As shown, 346 DEMs were detected in the serum samples of the high antibody group and the low antibody group on the 90th day after the third dose of vaccination, of which 169 DEMs were upregulated in the high antibody group relative to the low antibody group, and 177 DEMs were downregulated in the high antibody group relative to the low antibody group.

[0087] Intersection analysis was performed on serum metabolites at the 28th and 90th time points, and 106 DEMs with intersections were obtained. These 106 DEMs can be considered as serum basal metabolites that are stably present in the serum of the subjects, and may serve as serum metabolites that reflect the characteristics of the antibody response to the new crown inactivated vaccine.

[0088] MESA metabolite set enrichment analysis was performed on 106 DEMs, and KEGG enrichment analysis enriched 11 metabolic pathways. Figure 5 As shown, 4-hydroxyphenylpyruvate (4-HPP), which is involved in the biosynthesis pathway of ubiquinone and other terpenoid quinones and the tyrosine metabolic pathway, had a lower serum abundance in the high antibody group than in the low antibody group on day 28 after the third dose of vaccination, while it had a higher serum abundance in the high antibody group than in the low antibody group on day 90. Figure 6 As shown, isopentenol, which is involved in the riboflavin metabolic pathway, was consistent with the serum abundance results of 4-HPP. On the 28th day after the third dose of vaccination, the serum abundance of the high antibody group was lower than that of the low antibody group, while on the 90th day, the serum abundance of the high antibody group was higher than that of the low antibody group. Figure 5 and Figure 6 In the figure, HIGH indicates the high antibody group, LOW indicates the low antibody group, and the box plots are the maximum value, 75% site value, median value, 25% site value, and minimum value from top to bottom. The Kolmogorov-Smirnov (KS) test was used for comparison between groups.

[0089] Analysis of the change trend of serum abundance of isopentenol and 4-HPP in serum samples of subjects in the high antibody group and the low antibody group at two time points: 28 days and 90 days after the third dose of vaccination. Figure 7 and Figure 8 As shown, prenol and 4-HPP showed a significant downward trend from day 90 to day 28 in the high antibody group, while they showed an increasing trend in the low antibody group. Figure 7 and Figure 8 A point in represents the test result of a subject.

[0090] 3. Results Analysis

[0091] like Fig. 9 As shown in Figure 1, 4-HPP is a ketoacid involved in the tyrosine catabolic pathway. 4-HPP is a product of (R)-4-hydroxyphenyllactate dehydrogenase and is formed during tyrosine metabolism. Tyrosine aminotransferase (TAT) catalyzes the conversion of tyrosine to 4-HPP. Oxidative phosphorylation or other reductases catalyze the conversion of 4-HPP to 4-hydroxybutyrate (4-HB), a process involving the participation of reduced coenzyme (NADH), which provides hydrogen ions and electrons to reduce 4-HPP.

[0092] 3-Methylbut-1-en-1-ol (isopentenol) and 3-methylbut-2-en-1-ol (prenol) are isomers of each other, both are isopentenols, and the two can be converted into each other.

[0093]

[0094] like Fig. 9 As shown in Figure 1, isopentenol can react with pyrophosphate (ppi) to generate dimethylallyl pyrophosphate (DMAPP) in the mevalonate pathway, and then synthesize polyprenyl pyrophosphate (Polyprenyl pyrophosphate) through a series of enzymatic reactions. Polyprenyl pyrophosphate is then combined with 4-HB to synthesize coenzyme Q10 (Ubiquinone / Coenzyme Q10, CoQ10).

[0095] See also Figure 1 and Figure 2 Among the test time points from the 28th day to the 300th day after the third dose of the vaccine, the antibody sVNT titer was the highest on the 28th day, then declined rapidly within the next 90 days, and entered a more stable stage after 90 days. Figure 5-Figure 9It can be seen that on the 28th day after the third dose of vaccination, in the high antibody group, the levels of serum isopentenol and 4-HPP were lower than those in the low antibody group, indicating that the utilization rate of isopentenol and 4-HPP at this stage was high and the consumption was large, which promoted the large-scale production of coenzyme Q10. In the low antibody group, on the 28th day, the consumption of serum isopentenol and 4-HPP was small, the level of synthetic coenzyme Q10 was lower than that in the high antibody group, and the serum COVID-19 antibody level was low. From the 28th day to the 90th day, the antibody sVNT titer decayed rapidly, the utilization rate of isopentenol and 4-HPP decreased, the consumption decreased, and the compensatory accumulation of isopentenol and 4-HPP in the body made the serum abundance of isopentenol and 4-HPP in the high antibody group higher than that in the low antibody group on the 90th day.

[0096] Coenzyme Q10 plays a variety of cellular functions in potential cellular functions related to the immune system. First, Coenzyme Q10 acts as an electron carrier in the mitochondrial oxidative respiratory chain, transporting electrons from class I complex NADH and class II complex to class III complex (ubiquinone-cytochrome c reductase), and produces ATP in the process. Reduced expression of Coenzyme Q10 synthase can inhibit ATP production and lead to organ dysfunction, and supplementation of Coenzyme Q10 can reverse this phenomenon. Energy consumption during the immune response is accompanied by the supply of Coenzyme Q10, so that various immune cells of the immune system can play their role. Secondly, Coenzyme Q10 also has the function of a fat-soluble antioxidant, mediating the ferroptosis inhibition of ferroptosis inhibitory protein 1, making cell membranes and circulating lipoproteins resistant to oxidative damage induced by free radicals. The antioxidant function of Coenzyme Q10 is attributed to its fully reduced ubiquinone (ubiquinol) that can capture lipid peroxyl radicals that mediate lipid peroxidation. Coenzyme Q10 can reduce the production of free radicals and protect immune cells from oxidative damage. Thirdly, Coenzyme Q10 exerts anti-inflammatory effects through NF-κB1-dependent gene expression, and may play a role in diseases such as cardiovascular disease and mitochondrial disease that require the inhibition of pro-inflammatory cytokine release, and has a certain immunomodulatory effect. Finally, Coenzyme Q10 helps to enhance immune memory. For example, memory CD8+T cells deploy a series of adaptability in tissue residence, including increased activity of the Coenzyme Q10 transcription factor Sterol Regulatory Element Binding Protein 2 (SREBP2). By deleting or overexpressing enzymes in the Coenzyme Q10 biosynthetic pathway, the synthesis of Coenzyme Q10 is enhanced, which can promote mitochondrial respiration and enhance memory T cell formation and anti-tumor immunity after viral infection. Therefore, Coenzyme Q10 can help enhance the host's antibody response function in vaccine immune response by assisting ATP synthesis to increase energy supply, protecting immune cells from oxidative damage, inhibiting inflammatory response to regulate adaptive immune response, and enhancing immune memory function.

[0097] In this way, increasing the levels of isopentenol and 4-HPP related to the synthesis of coenzyme Q10 in the body, or supplementing exogenous isopentenol and 4-HPP, can improve the immune response effect of the vaccine by increasing the synthesis of coenzyme Q10.

[0098] The logistic diagnostic model was used to establish an equation for the change in serum abundance of metabolites isopentenol and 4-hydroxyphenylpyruvic acid in the high antibody group and the low antibody group from day 28 to day 90. The equation is y = β1 + β2△C 异戊烯醇 +β3△C 4-羟基苯丙酮酸 , where y represents the antibody high group or antibody low group, △C represents the abundance of serum metabolites on day 90 minus the abundance of serum metabolites on day 28, and β1, β2, and β3 represent the parameters automatically generated by the diagnostic model. Fig.10 As shown, the area under the ROC curve was 0.9041; the 95% confidence interval was 0.8565 to 0.9517, P < 0.0001, indicating that the diagnostic model has a high degree of discrimination for the antibody response after vaccination, and can be used to judge the body's immune response level after immunization, thereby inferring the prognosis of vaccine immunity or vaccine treatment.

[0099] Embodiment 3

[0100] This example uses serum prenol abundance as an example to study the correlation between the immune consumption of serum prenol and the concentration of human serum apolipoprotein A1 (Apolipoprotein-A1, APO-A1). Immune consumption refers to the consumption of prenol in the immune response process after the body is vaccinated.

[0101] APO-A1 is considered a functional protein. Most of APO-A1 in serum is bound to high-density lipoprotein (HDL). As a cholesterol scavenger, it is considered an important factor in preventing thrombotic diseases. At the same time, APO-A1 also has certain immunoregulatory functions. It can mediate the release of extracellular IPP from DCs together with ATP-binding cassette transporter 1 (ABCA1), thereby activating Vg9Vd2 T cells, inducing their proliferation, and enhancing immune responses.

[0102] The APO-A1 concentration in the serum samples of the subjects in the high antibody group and the low antibody group was detected by immunoturbidimetry (Apolipoprotein A1 Assay Kit from Abbott). Fig.11, serum APO-A1 concentration (g / L) on day 28 after the third dose of vaccination was negatively correlated with serum prenol abundance (r represents Pearson correlation coefficient, r = -0.17, p = 0.0318). Therefore, subjects in the high antibody group and the low antibody group had low serum prenol abundance and high serum APO-A1 concentration; subjects in the high antibody group and the low antibody group had high serum prenol abundance and low serum APO-A1 concentration.

[0103] Thus, 28 days after the third dose of the vaccine, the immune response is active, and a large amount of isopentenol is consumed to produce more APO-A1, enhancing the antigen presentation ability of APC; at the same time, the level of coenzyme Q10 is increased, the mitochondrial oxidative phosphorylation function is enhanced, and ATP synthesis is increased to provide energy for antibody production, thereby enhancing the antibody response of the vaccine. In other words, the immune consumption of isopentenol in the immune response is positively correlated with the level of APO-A1 in the serum, and the level of APO-A1 in the serum can be detected to indicate the immune consumption of isopentenol in the immune response.

[0104] Embodiment 4

[0105] This example takes prenol as an example to study the enhancing effect of prenol on the immune response of the vaccine.

[0106] BALB / c 7-week-old female mice were randomly divided into 4 groups, 5 mice in each group. The day of the first dose of vaccine was set as day 0, and the dosing method of mice in each group was as follows:

[0107] Inactivated vaccine + metabolite group (BBIBP + prenol): Starting from day -3 (3 days before the first dose of vaccine), mice began to be intraperitoneally injected with 200 μL of prenol (dosing concentration 50 mg / kg / d), and then administered once every two days until day 42; at the same time, a dose of 2.6U (200 μL) of the commercialized COVID-19 inactivated vaccine BBIBP-CorV produced by Beijing Institute of Biological Products, Sinopharm Group was administered on days 0, 14, and 28, respectively.

[0108] Inactivated vaccine group (BBIBP): Starting from day -3, mice began to be intraperitoneally injected with 200 μL of PBS, and then the drug was administered once every two days until day 42; at the same time, a dose of 2.6U (200 μL) of the commercialized COVID-19 inactivated vaccine BBIBP-CorV produced by Beijing Institute of Biological Products, Sinopharm Group was administered on days 0, 14, and 28 respectively.

[0109] Metabolite group (prenol): Starting from day -3, mice began to be intraperitoneally injected with 200 μL of prenol (dosing concentration 50 mg / kg / d), and then administered once every two days until day 42; at the same time, 200 μL of PBS were inoculated on days 0, 14, and 28, respectively.

[0110] Control group (PBS): Starting from day -3, mice were intraperitoneally injected with 200 μL of PBS, and then administered once every two days until day 42; at the same time, 200 μL of PBS was inoculated on days 0, 14, and 28, respectively.

[0111] Blood samples were collected from the four groups of mice on days 0, 14, 28, and 42, and the serum APO-A1 concentration was detected by enzyme-linked immunosorbent assay (ELISA), and the sVNT titer of COVID-19 antibodies was detected by alternative virus neutralization test. Fig.12 and Fig.13 As shown, the serum COVID-19 antibody sVNT titer of the inactivated vaccine + metabolite group on the 14th day after the third dose (the 42nd day after the first dose) was 2.44 times that of the inactivated vaccine group, significantly higher than that of the inactivated vaccine group (p = 0.0161), and the serum APO-A1 concentration of the inactivated vaccine + metabolite group on the 42nd day was significantly higher than that of the inactivated vaccine group (p = 0.0317). In other words, when mice were supplemented with exogenous metabolites of prenyl alcohol during vaccination, the serum COVID-19 antibody sVNT titer increased, and the serum APO-A1 concentration increased.

[0112] This indicates that supplementing exogenous prenol can increase the immune consumption of prenol, thereby increasing the concentration of APO-A1 in serum. At the same time, the increased consumption of prenol increased the antibody level in mouse serum, indicating that supplementing exogenous prenol can improve the immune response effect of the vaccine.

[0113] Embodiment 5

[0114] This example uses prenol as an example to study the inhibitory effect of prenol on the growth of B-cell lymphoma in mice.

[0115] The B-cell lymphoma mouse tumor model (tumor-bearing model mouse) was constructed by subcutaneously bearing tumors with the A20 cell line in BALB / c mice. The method for constructing tumor-bearing model mice is as follows: On day 0, the A20 cell line was subcutaneously injected into the right armpit of the BALB / c mice. After 3 days of feeding, the long and short diameters of the subcutaneous tumor of each mouse were measured, and the mice were weighed; thereafter, the tumor volume was measured every 3 days, and the mice were weighed and recorded every week. On the 10th day of tumor bearing, the tumor-bearing model mice with successful B-cell lymphoma mouse tumor model were randomly divided into four groups according to the growth of the tumor. The tumor-bearing model mice in each group were administered with drugs starting from the 10th day of tumor bearing. The specific administration method is as follows:

[0116] Metabolic group (prenol): prenol was administered once every two days at a dose of 50 mg / kg / d for a total of 5 times.

[0117] Anti-PD1 antibody treatment group (anti-PD1): Anti-PD1 antibody was administered once every two days, with a dose of 100 μg / mouse, for a total of 5 times.

[0118] Metabolite combined with anti-PD1 antibody treatment group (prenol+anti-PD1): anti-PD1 antibody and prenol were administered once every two days, with a dose of 100 μg / animal anti-PD1 antibody and 50 mg / kg / d prenol, for a total of 5 times.

[0119] Control group (PBS): PBS was administered once every two days for a total of 5 times.

[0120] The anti-PD1 antibody was InVivoMAb anti-mouse PD-1 (CD279), from Biocell, USA. Starting from day 0 of tumor bearing, the long diameter L and short diameter D of the tumor were measured every 3 days to calculate the tumor volume. The calculation formula of tumor volume is as follows:

[0121] Tumor volume = 0.52 × L × D 2

[0122] See also Fig.14 Repeated measures variance was used for analysis of variance among the groups, where * indicates a p value less than 0.05 and **** indicates a p value less than 0.0001. Fig.14 It can be seen that the tumor growth rate of the metabolite group was significantly lower than that of the control group (p < 0.0001), indicating that the addition of exogenous isopentanol can significantly reduce the growth rate of tumors. The tumor growth rate of the metabolite combined with anti-PD1 antibody treatment group was also significantly lower than that of the anti-PD1 antibody treatment group (p = 0.0177), indicating that isopentanol can cooperate with anti-PD1 antibodies to inhibit the growth of mouse tumors.

[0123] Thus, on the one hand, isopentanol can be used to prepare a pharmaceutical preparation, which can be used alone or in combination with other anti-tumor methods or anti-tumor products. For example, it can be used in combination with other chemotherapy drugs or in combination with radiotherapy. On the other hand, isopentanol can also be used as an adjuvant and added to tumor therapeutic vaccines to enhance the immune response of tumor therapeutic vaccines and enhance the anti-tumor effect.

[0124] It should be noted that this embodiment uses isopentanol as an example for research. According to the results of the metabolomics study in Example 2, both 4-HPP and isopentanol are DEMs obtained in the KEGG enrichment analysis, and both have lower serum abundance in the high antibody group than in the low antibody group on the 28th day after the third dose of immunization, and higher serum abundance in the high antibody group than in the low antibody group on the 90th day. At the same time, 4-HPP and isopentanol are both intermediates in the coenzyme Q10 synthesis pathway. Therefore, 4-HPP also has the effect of improving immune response, and can be used alone or in combination with isopentanol to prepare a pharmaceutical preparation for improving immune response.

[0125] Those skilled in the art will readily appreciate other embodiments of the present disclosure after considering the specification and practicing the invention disclosed herein. This application is intended to cover any modification, use or adaptation of the present disclosure, which follows the general principles of the present disclosure and includes common knowledge or customary techniques in the art that are not disclosed in the present disclosure. The specification and examples are intended to be exemplary only, and the true scope and spirit of the present disclosure are indicated by the appended claims.

Claims

1. A preparation for enhancing immune response, characterized in that Includes at least one of isopentenol and 4-hydroxyphenylpyruvate.

2. The preparation for enhancing immune response according to claim 1, characterized in that The preparation is an injection or an oral preparation.

3. The preparation for enhancing immune response according to claim 1 or 2, characterized in that The prenyl alcohols include 3-methylbut-1-en-1-ol and 3-methylbut-2-en-1-ol.

4. Use of the preparation according to any one of claims 1 to 3 in the preparation of combined immune products.

5. The use according to claim 3, characterized in that: The combined immunity product achieves combined immunity by the following method: S10. Obtaining a combined immune product, wherein the combined immune product includes the preparation and the vaccine; S20, administration before vaccine immunization: before vaccine immunization, the first preset process is used to complete the administration of the preparation described in step S10; Administration during vaccine immunization: the administration of the vaccine described in step S10 is completed by using the second preset process, and at the same time, the administration of the preparation described in step S10 is completed by using the third preset process during the vaccine immunization process; Administration after vaccine immunization: After the last vaccine immunization, the fourth preset process is used to complete the administration of the preparation described in step S10.

6. Use of the preparation according to any one of claims 1 to 3 as an adjuvant in the preparation of a composite vaccine.

7. The use according to claim 6, characterized in that: The immunization method of the composite vaccine comprises the following steps: S1. Obtaining a composite vaccine, wherein the composite vaccine comprises a vaccine and the preparation; S2. Complete the immunization administration of the combined vaccine according to the fifth preset process.

8. Use of the preparation according to any one of claims 1 to 3 in the preparation of anti-tumor products.

9. The use according to claim 8, characterized in that: The anti-tumor product is an anti-tumor compound preparation or a combined anti-tumor composition; The anti-tumor composite preparation comprises the preparation and an anti-tumor drug, and is used to allow the preparation and the anti-tumor drug to be administered simultaneously; The combined anti-tumor composition comprises the preparation and an anti-tumor drug, and is used to allow the preparation and the anti-tumor drug to be used in combination and administered separately.

10. Application of isopentenol and / or 4-hydroxyphenylpyruvate as biomarkers in monitoring the body's immune response level.

11. Application of isopentenol and / or 4-hydroxyphenylpyruvate as biomarkers in screening drugs that regulate metabolic pathways; The drug for regulating metabolic pathways is used to increase the synthesis of isopentenol and / or 4-hydroxyphenylpyruvate, or the drug for regulating metabolic pathways is used to reduce the degradation or excretion of isopentenol and / or 4-hydroxyphenylpyruvate.

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